JP2002017386A - Method for producing indole-3-carboxylic acid derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing an indole-3-carboxylic acid derivative useful as an intermediate for synthesizing medicals/ agrochemicals. SOLUTION: This method comprises subjecting an indole derivative to the action of microorganismal cells or a treated product thereof having the ability to convert the above derivative to the corresponding indole-3-carboxylic acid derivative and collecting the objective indole-3-carboxylic acid derivative formed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an indole-3-carboxylic acid derivative from an indole derivative by the action of an enzyme. These indole-3-
Carboxylic acid derivatives are useful as raw materials for various medical and agricultural chemicals.

[0002]

BACKGROUND ART Enzymatic decarboxylation in living bodies has been studied in detail for amino acids and α-keto acids, and their enzymatic properties have been elucidated. However, there are many unclear points about non-oxidative decarboxylase of aromatic carboxylic acids. As a non-oxidative decarboxylation reaction of an aromatic carboxylic acid by a microorganism, a reaction of converting hydroxybenzoic acid to phenol is known (Microb. Ecol.,
20 , 103, 1990). In addition, Citronact
It is known that bacteria belonging to the genus er decarboxylate gallic acid and produce and accumulate pyrogallol (Agric. Biol.
Chem. , 46 , 2539, 1982).

[0003] For microorganisms that catalyze the fixation of carbonic acid to aromatic compounds, 4-
Conversion to hydroxybenzoic acid is known for bacteria of the genus Pseudomonas (Arch. Microbiol., 148 , 213, 1987).
Clostrid is a carbon dioxide fixation by decarboxylation and reverse reaction of 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid
It has been shown that it is catalyzed by an enzyme derived from the genus ium (Appl. Environ. Microbiol., 60 , 4182, 1994). Also, Ba
It has been found that pyrrole-2-carboxylic acid decarboxylase from the genus cillus performs carbonic acid fixation on pyrrole to synthesize pyrrole-2-carboxylic acid (Eur. J. Bio.
Chem., 253 , 480, 1998). Furthermore, a similar activity has been found in an enzyme derived from Serratia (Japanese Patent Application No. 11-539).
issue). On the other hand, there is no report on enzymatic decarboxylation reaction and its reverse reaction, carbonic acid fixation reaction, for indole derivatives which are polycyclic compounds.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an industrially advantageous production method of an indole-3-carboxylic acid derivative useful as an intermediate for synthesis of medicinal and agricultural chemicals by an enzymatic carbon fixation reaction. .

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies on enzymatic decarboxylation of an indole derivative and carbonic acid fixation, which is the reverse reaction, and as a result, have obtained a method for immobilizing carbonic acid on an indole derivative in a specific manner. The discovery of the presence of an enzyme that catalyzes the reaction led to the completion of the present invention.
That is, the present invention relates to a method for producing an indole-3-carboxylic acid derivative, wherein the compound represented by the general formula (III)

Embedded image (Wherein X1 to X6 represent a hydrogen atom or a substituent) by converting an indole derivative represented by the general formula (IV) in the presence of a carbonate ion from an indole derivative:

Embedded image (Wherein X1 to X6 represent a hydrogen atom or a substituent), an enzyme having an ability to catalyze the synthesis of an indole-3-carboxylic acid derivative, an enzyme-immobilized product, a microorganism, a cell culture solution, or a microorganism A method for producing an indole-3-carboxylic acid derivative, which comprises collecting a produced indole-3-carboxylic acid derivative by treating a treated body.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the general formulas (I) and (II), X1 to X6 represent a hydrogen atom or a substituent, and are not particularly limited as long as the reaction is not inhibited. Specifically, in the case of X1, an alkyl group,
Substituents such as an alkenyl group, a cycloalkyl group, an aralkyl group, an acyl group, an amino group, a hydroxyl group, a halogen group, an alkoxyl group, an oxycarbonyl group, and a silyl group, and substituents derived therefrom are represented by X2 to X6. In the case, substituents such as alkyl group, alkenyl group, cycloalkyl group, aralkyl group, acyl group, amino group, hydroxyl group, halogen group, nitrile group, nitro group, carboxyl group, alkoxyl group, oxycarbonyl group, silyl group and the like And the like.

[0007] The indole derivative used as a raw material can be synthesized by a known method. The enzyme, the enzyme-immobilized product, the microorganism, the culture of the bacterial cell, or the treated product of the bacterial cell used in the present invention is obtained by converting indole derivative to indole-3-
The type and origin of the carboxylic acid derivative are not limited as long as it has the ability to catalyze the carbonic acid fixing reaction to the carboxylic acid derivative.

The enzyme or enzyme-immobilized product that catalyzes this reaction includes, for example, a crude enzyme or a purified enzyme isolated from a microorganism having the ability to catalyze a carbonic acid fixation reaction from an indole derivative to an indole-3-carboxylic acid derivative. Can be used. Such microorganisms include:
Although there is no particular limitation, for example, Arthroba
Microorganisms belonging to the genus cter, the genus Gibberella, the genus Fusarium and the like can be mentioned.

As microorganisms belonging to the genus Arthrobacter,
Arthrobacter nicotiance FI 1612 (FERM P-17955)
However, Gibberella is a microorganism belonging to the genus Gibberella.
Examples of microorganisms belonging to the genus Fusarium include fujikuroi IFO 6605 and Fusarium subglutinans FI31 (FERMP-17954). Arthrobacter nicotiance FI 1612 and F
usarium subglutinans FI 31 is newly isolated from the soil by the present inventors, and has been deposited with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology, under the above deposit number, and has the following biological properties. It is on the street.

Arthrobacter nicotiance FI 1612 form; Bacillus Gram stain; + spores;-oxygen demand; aerobic motility;-catalase; + peptidoglycan type; A4α, L-Lys-L-.
No meso-diaminopimelic acid was found in the hydrolyzate of Ala-L-Glu cells; no production of acid or gas from glucose was found. Furthermore, based on the similarity of the 16S rDNA sequence, Arthr
Conclusion with obacter nicotiance.

Properties of Fusarium subglutinans FI 31 colony: A colony having a diameter of 30 mm is formed in an oatmeal medium at 25 ° C. for 3 days, and the aerial mycelium becomes as high as about 3 mm. Agar has a tan color. Conidia clusters are present in the orange conidium on the agar surface. Consists of many dark sclerotia. Morphology: ball-shaped conidia sticky to aerial mycelia are typically 50 μm long commonly seen or growing stalk-shaped phialides, 8-20 μm long elliptical / comboid elliptical conidia. It is formed. The large conidium of the conidial locus is composed of a tufted conidiophore and a tubular incisor (conidia forming cells), and is 25 μm in length. Conidia have about 3-4 septa. Distinct podocytes are indistinct and have bent apical cells of 30-35 x 3.5 μm. No chlamydospores are present.

Gibberella fujikuroi IFO 6605 is known and can be easily obtained from the Fermentation Research Institute. It is also possible to use genetically engineered microorganisms in which enzyme genes isolated from these microorganisms have been introduced into various host vector systems.

Furthermore, not only the crude enzyme or the purified enzyme isolated from the microorganism as described above, but also a culture obtained by culturing the microorganism in a culture medium as it is or by collecting the culture by centrifugation or the like. Culture supernatant obtained by bacterial operation,
Indole-3- in the presence of cells or processed cells
Carboxylic acid derivatives can also be produced. Examples of the treated cells include cells treated with acetone, toluene, etc., crushed cells, cell-free extracts obtained by crushing cells, and the like.

In the present invention, these enzymes, enzyme-immobilized products, microorganisms, bacterial cell cultures, or treated bacterial cells are usually
Although a kind is used, it is also possible to mix and use two or more kinds having the same ability.

In the production method of the present invention, when the enzyme is subjected to the reaction, the form of use is not particularly limited as long as the enzyme shows activity, and the enzyme may be immobilized on a suitable carrier before use. By using the enzyme immobilized, the separation and recovery of the indole-3-carboxylic acid derivative and the enzyme after the reaction is completed, and the enzyme can be reused.

In the present invention, as a medium for culturing these microorganisms, any medium can be used as long as these microorganisms can grow. Examples of the carbon source include sugars such as glucose, sucrose and maltose, organic acids such as acetic acid, citric acid and fumaric acid and salts thereof, and alcohols such as ethanol and glycerol. As a nitrogen source, for example, peptone,
In addition to general natural nitrogen sources such as meat extract, yeast extract and amino acids, various inorganic and organic acid ammonium salts can be used.
In addition, inorganic salts, trace metal salts, vitamins and the like are added as needed. Also, to obtain high enzyme activity,
For example, it is also effective to add a compound having an indole skeleton, such as indole, indole-3-carboxylic acid, tryptophan, and 3-cyanoindole, or a compound having a carboxylic acid as a substituent, to the medium as an inducer of enzyme production.

The cultivation may be carried out according to a conventional method. For example, the cells are cultured aerobically at a pH of 4 to 10 and a temperature of 15 to 40 ° C. for 6 to 96 hours. In some cases, high enzymatic activity can be obtained by culturing the cells in a static culture in the same manner.

In the present invention, the production of the indole-3-carboxylic acid derivative by the carbonic acid fixation reaction can be carried out by the following method. The reaction can be carried out by contacting the indole derivative with the above enzyme, immobilized enzyme, microorganism, cell culture, or treated cell in a reaction solvent such as water or a buffer solution in the presence of carbonate ions. Then, the reaction is performed while controlling the reaction temperature, the internal pressure in the reaction system, and if necessary, the pH of the reaction solution. In some cases, during the reaction, a carbonate ion or an indole derivative may be added to continue the reaction.

The concentration of the substrate in the reaction solution is not particularly limited between 0.01 and 50% by mass, but it is not limited to 0.1 in consideration of productivity and the like.
It is preferably carried out at a concentration of 1 to 30% by weight. As a source of carbonate ions, carbon dioxide or sodium,
Potassium, carbonates such as ammonium and the like, in particular,
Potassium hydrogen carbonate and sodium hydrogen carbonate are effective. Since this reaction is an equilibrium reaction, the reaction yield can be further increased by conducting the reaction under carbon dioxide gas pressure in addition to these salts.

The concentration of microorganisms and the like in the reaction solution is usually 0.1.
It is from 0.01 to 20% by mass, preferably from 0.01 to 10% by mass.

The pH of the reaction solution is determined comprehensively in consideration of the optimum pH of the enzyme to be used, the solubility of carbonate ions, and the like, and is not particularly limited, but is generally in the range of pH 4 to 11. Has a pH of 5 to 9. In addition, the pH changes as the reaction proceeds.In this case, an appropriate neutralizing agent is added to optimize the pH.
It is desirable to adjust to pH.

The reaction temperature is preferably from 0 to 60 ° C, and from 5 to 5 ° C.
0 ° C. is more preferred.

As the reaction solvent, an aqueous medium such as ion-exchanged water or a buffer is usually used, but the reaction can also be carried out in a system containing an organic solvent or a surfactant to promote the dissolution of the indole derivative. Examples of the organic solvent include alcohol solvents such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butyl alcohol, and t-amyl alcohol; and aliphatic hydrocarbon solvents such as pentane, hexane, heptane, and octane. , Benzene, toluene, xylene and other aromatic hydrocarbon solvents, methylene chloride, chloroform, carbon tetrachloride, dichloroethane and other halogenated hydrocarbon solvents,
Ether solvents such as diethyl ether, diisopropyl ether, tetrahydrofuran and dioxane; ester solvents such as ethyl acetate, propyl acetate and butyl acetate; ketone solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone; other acetonitrile, N, N-dimethyl Formamide and the like can be appropriately used. Examples of the surfactant include anionic surfactants such as alkylbenzene sulfonate and alkyl sulfate, cationic surfactants such as alkylpyridinium salt and dodecyltrimethylammonium chloride, polyoxyethylene alkyl (phenyl) ether, and polyoxyethylene. Alkyl (phenyl) ester, sorbitan fatty acid ester (span-based surfactant), polyoxyethylene glycol sorbitan alkyl ester (tween-based surfactant), polyoxyethylene glycol pt-octylphenyl ether (triton-based surfactant), Nonionic surfactants such as sugar fatty acid esters and amphoteric surfactants such as N-alkyl-N, N-dimethylammonium betaine, lecithin, phosphatidylethanolamine, lysolecithin and the like are appropriately used. You can use.

Further, it is possible to add two or more of these organic solvents or surfactants to a solution having a solubility equal to or higher than the solubility in water to carry out the reaction in a two-layer system. By allowing an organic solvent to coexist in the reaction system,
Selectivity, conversion, yield, etc. are often improved. The reaction time is generally 1 hour to 1 week, preferably 1 to 72 hours, and it is preferable to select reaction conditions under which the reaction is completed.

The above-mentioned substrate or carbonate ion concentration, enzyme concentration, pH, temperature, solvent, reaction time, and other reaction conditions are determined in consideration of the reaction yield and the like under such conditions. It is desirable to appropriately select conditions under which the most carboxylic acid derivative can be collected.

The target substance can be isolated from the mixed solution after the completion of the carbonic acid fixation reaction by a known method such as concentration, extraction, column separation, crystallization, etc. after removing the bacteria.

[0027]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to examples, but the scope of the present invention is not limited to the examples.

Example 1 Indole-3-carboxylic acid 2.0 g / l, yeast extract 1.0 g / l, (NH4)
2HPO4 2.0g / l, MgSO4.7H2O
30 ml of medium consisting of 0.5 g / l, vitamin mixture 5 ml / l and metal salt mixture 5 ml / l (pH 7.0)
Was dispensed into a 500 ml Sakaguchi flask at 121 ° C., 15
After heat sterilization for minutes, the strains shown in Table 1 were inoculated at 28 ° C.
For 28 hours with shaking. The vitamin mixture was 100 mg / l biotin and 20 mg calcium pantothenate.
/ L, inositol 100mg / l, nicotinic acid 20m
g / l, pyridoxine hydrochloride 20 mg / l, p-aminobenzoic acid 10 mg / l, riboflavin 10 mg / l, folic acid 0.5 mg / l, and the metal salt mixture was H3BO.
4 300 mg / l, CaCl2 400 mg / l, C
uSO4.7H2O 40mg / l, KI 100mg
/ L, FeSO4.7H2O 200mg / l, MnS
O4.7H2O 400mg / l, H2MoO4.2H
It consists of 200 mg / l of 20 and 10 ml / l of concentrated hydrochloric acid.

After completion of the culture, the cells are collected by centrifugation,
After washing with the same amount of 0.85% NaCl solution as the culture solution,
The cells were suspended in 1.5 ml of the same solution. 100 mM indole solution (dissolve indole in 40% methanol,
Prepared 100 mM solution) 0.4 ml, 0.5 M
0.4 ml of potassium phosphate buffer (pH 6.0), K
After adding 0.6 mmol of HCO3 and 1.0 ml of the cell suspension shown in Table 1, respectively, and adjusting the total amount to 2 ml,
It was sealed and reacted at 20 ° C. for 6 hours. After removing the bacteria from the reaction solution by centrifugation, indole-3-carboxylic acid was quantified by high performance liquid chromatography. Table 1 shows the results.

Table 1.

Example 2 Maltose 10 g / l, meat extract 5 g / l, indole-3-carboxylic acid 1.5 g / l
l, yeast extract 0.5 g / l, MgSO4.7H2O
30 ml of a medium consisting of 0.5 g / l, 1 g / l of K2HPO4 and 5 ml / l of a metal salt mixture (pH 7.0)
Was dispensed into a 500 ml Sakaguchi flask at 121 ° C., 15
After heat sterilization for 1 minute, the Arthrobacter nicotiance FI 161
2 was inoculated, and cultured with shaking at 28 ° C. for 28 hours.

After completion of the culture, the cells were collected by centrifugation,
After washing with the same amount of 0.85% NaCl solution as the culture solution,
The cells were suspended in 1.5 ml of the same solution.

0.4 ml of a 100 mM indole solution (prepared by dissolving indole in 40% methanol to prepare a 100 mM solution) and 0.5 M potassium phosphate buffer (p
H6.0) 0.4 ml, KHCO3 0.6 mmol
And add 1.0 ml of Arthrobacter nicotiance FI 1612 cell suspension, adjust the total volume to 2 ml, stopper tightly,
The reaction was performed at 20 ° C. for 6 hours. After removing bacteria from the reaction solution by centrifugation, indole-
As a result of the quantification of 3-carboxylic acid, formation of 7.0 mmol / L of indole-3-carboxylic acid was confirmed.

Example 3 As in Example 2, Arthrobact
er nicotiance FI 1612 was cultured on a 5-fold scale and reacted on a 5-fold scale (reaction solution: 10 ml) under the same conditions.
After 6 hours, indole-3-carboxylic acid 10 mmol /
L formation was observed.

This reaction was repeated 15 times, and the product indole-3-carboxylic acid was isolated. That is, after removing bacteria by centrifugation from the reaction solution for 15 times, carbon dioxide was distilled off under reduced pressure. Dowex 1 × 2 (O
(H ^- form), washed with water and 0.05N acetic acid, and eluted with 2N acetic acid. The eluate was extracted three times with ethyl acetate and concentrated to dryness. Recrystallization from ethyl acetate / n-hexane gave 106 mg of indole-3-carboxylic acid. ^The structure was confirmed by ¹ H-NMR and ¹³ C-NMR.

Example 4 Cell suspensions shown in Table 2 were prepared in the same manner as in Example 1 and subsequently reacted in the same manner except that 2-methylindole was added instead of indole. After removing the bacteria from the reaction solution by centrifugation, 2-methylindole-3-carboxylic acid was quantified by high performance liquid chromatography.

Table 2.

According to the present invention, it is possible to produce an indole-3-carboxylic acid derivative useful as an intermediate for synthesizing a medicinal and agricultural chemical by a very mild enzymatic method.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) (C12P 17/10 (C12P 17/10 C12R 1:06) C12R 1:06) (72) Inventor Tetsuji Nakamura 10-1 Daikoku-cho, Tsurumi-ku, Yokohama-shi, Kanagawa F-term (reference) in Chemical Development Laboratory, Mitsubishi Rayon Co., Ltd. 4B064 AE48 CA02 CA05 CB30 CD12 DA01

Claims (2)

[Claims] 1. A method for producing an indole-3-carboxylic acid derivative, comprising the steps of: preparing a compound represented by the general formula (I): (Wherein X1 to X6 each represent a hydrogen atom or a substituent) by converting an indole derivative represented by the general formula (II) in the presence of a carbonate ion: (Wherein X1 to X6 represent a hydrogen atom or a substituent), an enzyme having an ability to catalyze the synthesis of an indole-3-carboxylic acid derivative, an enzyme-immobilized product, a microorganism, a cell culture solution, or a microorganism A method for producing an indole-3-carboxylic acid derivative, which comprises reacting a body treated product and collecting an indole-3-carboxylic acid derivative produced. 2. The indole derivative and the indole-3-carboxylic acid derivative represented by the general formulas (I) and (II), wherein X 1 and X 3 to X 6 are hydrogen atoms. For producing an indole-3-carboxylic acid derivative of the above.